Abstract

Allelic deletions of 9p including band 21–22 are common in various types of human carcinomas including breast cancer. Our previous cytogenetic studies had identified constitutional chromosomal changes in 9p23–24 in patients of a male-breast-cancer family and 9p23–24 alterations in a cell line established from a sporadic female breast cancer. To find out whether this genomic region is involved more frequently in alterations in sporadic breast cancers, we have surveyed 80 microdissected tumor samples for both loss of heterozygosity (LOH) and homozygous deletion at 22 microsatellite loci spanning 9p22 to 9p24 using fluorescent multiplex PCR. LOH at one or more loci was observed in 32 (40%) of these tumors. Homozygous deletion was detected in four cases. Eleven tumors had LOH at all of the informative loci analyzed, whereas 21 tumors showed partial-terminal or interstitial allelic loss of 9p. Deletion mapping identified two common regions of deletion: (a) 4 cM including D9S281 to D9S286; and (b) 1 cM including D9S1808 to D9S268.

Introduction

Breast cancer is the most common malignancy and the major cause of cancer-related deaths of women in industrialized countries
(1)
. Cytogenetic and molecular genetic analyses suggest that the accumulation of genetic changes including the activation of oncogenes as well as the inactivation of tumor suppressor genes are critical in the pathogenesis of breast cancer
(2, 3)
. Mutations in BRCA1 on 17q21 and BRCA2 on 13q12–13 are thought to be responsible for the majority of inherited breast cancers
(4, 5)
. In sporadic breast cancers, the expression of BRCA1 was found reduced or lost in a subset of high-grade tumors
(6)
, but mutations of BRCA1 and BRCA2 are rare
(7,
8,
9)
. Allelotype analyses of breast cancer have demonstrated frequent allele loss of chromosome arm 1p, 3p, 6p, 7q, 8p, 11p, 17p, 17q, and 18q, and it has been suggested that these deletions of genetic material indicate the loss of multiple tumor suppressor genes in breast cancer
(2, 3)
.

Alterations of 9p are common in many tumor types. Deletion studies have identified different regions encompassing 9p21–22 in various tumor types
(10,
11,
12,
13,
14,
15,
16,
17,
18,
19,
20,
21)
. We have previously reported the coexistence of constitutional chromosomal rearrangement in 9p23–24 and the mutation of BRCA2 in three brothers with breast cancer
(22)
, and the same 9p region was altered in breast cancer cell line COLO-824
(23)
. Previous LOH
3
studies of breast cancers had focused on 9p21–22, in particular the region encompassing the CDKN2 gene
(24,
25,
26,
27)
. To find out whether the distal portion of 9p is recurrently altered in sporadic breast cancers, we determined the allelic status of 22 microsatellite loci spanning 9p22 to 9p24 in 80 tumors. This study has revealed independent deletions in two common overlapping regions: (a) 4 cM including D9S281 to D9S286; and (b) 1 cM including D9S1808 to D9S268.

Materials and Methods

Materials and DNA Extraction.

Tumor materials were obtained from 80 patients with primary breast carcinoma at the time of primary surgery and were frozen immediately. Tumors were classified according to the tumor-node-metastasis (TNM) classification (Union Internationale Contre le Cancer). Tumor tissues were microdissected from 10-μm sections, and DNA was prepared as described previously
(13)
. Matched normal cell DNA was obtained either from peripheral blood collected at the time of surgery in EDTA or from sections of lymph nodes without tumor infiltration.

Fluorescent Multiplex PCR.

Microsatellite loci analyzed
4
are listed in Fig. 2
⇓
. The primers were purchased from Applied Biosystems (ABI, Weiterstadt, Germany). One primer of each pair was fluorescence-labeled at the 5′ end with one of the three dyes (6-FAM, JOE, and ROX), which made it possible to detect the amplified DNA individually. Multiplex PCR assays with two to six sets of primers were designed on the basis that coamplified products could be distinguished either by size or by color. PCR was performed in a 25-μl volume containing 50 ng of genomic DNA, 20 pmol of each primer, 150 μm each dNTP, 1.5 mm MgCl2, and 0.5 units of Taq polymerase (Promega, Madison, WI). Amplification was for 25–28 cycles consisting of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min followed by a final extension at 72°C for 8 min. The fluorescent products were mixed with internal standard-size markers (TAMRA 500, ABI) and fractionated by capillary-electrophoreses using an ABI 310. Data were collected automatically and analyzed by the Genescan 2.1 software (ABI), which provides quantification based on peak size and area. LOH and homozygous deletions were scored as described previously
(26, 28)
. Homozygous losses were verified by using different multiplex primer combinations.

Results

Normal and tumor DNA from 80 breast cancer patients were examined for allelic deletions at 22 microsatellite loci between 9p22–24. Allelic deletion (LOH and/or homozygous deletion) of at least one of these loci was detected in 32 (40%) of the 80 tumors analyzed (Fig. 1)
⇓
. The frequency of allelic deletion ranged from 19% at D9S1979 and to 38% at D9S1808. The highest frequency was observed at D9S1808 (38%) and D9S286 (37.4%). Homozygous deletion at D9S254 was detected in three tumors, whereas homozygous deletion at D9S268 was observed in one tumor. Tumor 60 exhibited homozygous deletion only at the D9S254 locus and had retained heterozygosity at all of the other informative loci. In 11 of the 32 tumors with 9p allelic loss, all of the informative loci were involved, which indicated the deletion of most of, or even the entire short arm of, chromosome 9. The remaining 21 tumors had partial-terminal or interstitial deletions (Fig. 2)
⇓
. Four tumors (T2, T47, T49, and T69) had two interstitial allelic deletions separated by a retained area.

Allelic deletion of 9p23–24 in tumor 47 detected by fluorescent multiplex PCR. The data were obtained with a DNA sequencer (ABI 310) and analyzed with Genescan 2.1 software. X-axis shows fragment size in bp. N, DNA from normal tissue; T, DNA from tumor tissue. Tumor DNA shows a LOH at D9S281 and D9S286, the loss of both alleles at D9S254, and the retention of heterozygosity at D9S288 and D9S256.

Deletion map of 9p in 21 sporadic breast carcinomas. The order and position of the markers are based on the integrated map and Généthon map of chromosome 9 from the human Genome Database. Vertical box, the region of deletion; solid bars on the right, the common regions of deletion A and B. ○, retention of heterozygosity; •, LOH; ▪, homozygous deletion; ◍, uninformative.

The deletion map of the 21 tumors with partial-terminal or interstitial loss revealed two common regions of deletion, one included D9S281 and D9S286 (approximately 4 cM, region A), and the other included D9S1808 and D9S268 (approximately 1 cM, region B). The border of region A was defined distally by tumors T5 and T46 and proximally by tumors T58 and T69. The border of region B was defined distally by tumors T46, T52, and T69 and proximally by T34 and T60. The frequencies of allele deletion in region A and B were 34% and 35%, respectively.

Discussion

Frequent LOH and homozygous deletions of 9p21–22 in 38 to 58% of breast cancers have been reported in deletion mapping studies with highly informative microsatellite markers
(24,
25,
26,
27)
. In the present study, we demonstrated allelic deletions of a more distal region, 9p23–24, in 40% of the breast cancers analyzed. Translocations and deletions of distal 9p have been detected in karyotypes of breast cancer by cytogenetic analysis
(29)
. Our previous cytogenetic studies have identified constitutional chromosomal rearrangements in 9p23–24 in patients of a male-breast-cancer family with mutation of BRCA2(22)
and in breast cancer cell line COLO-824
(23)
. In a LOH study with eight microsatellite loci of 9p, a frequently deleted region was mapped distal to the CDKN2 locus and extended to 9p23 in breast cancer
(24)
. However, the actual extent of the deleted region could not be determined clearly because of the low number of loci used. In the present study, we identified two common regions of deletion on 9p23 in breast cancer using a large set of microsatellite loci. The first region was between D9S281 and D9S286, and the second region was between D9S1808 and D9S268. Both regions of deletion were within, or in the vicinity of, the altered regions identified in the male breast cancer family and in the cell line COLO-824, which suggests that 9p is genetically unstable and may harbor genetic information involved in the tumorigenesis of breast cancer.

Allelic loss of 9p22–23 has been detected frequently in various types of human cancers including bladder cancer, lung cancer, melanoma, and neuroblastoma
(10,
11,
12,
13,
14,
15,
16,
17,
18,
19,
20,
21)
. The target region between D9S156 and D9S162 distal to the CDKN2 gene has been described in lung cancer
(13, 16)
, melanoma
(21)
, and squamous cell carcinoma of the esophagus
(20)
. In melanoma, a common region of loss extends distally from D9S171 to D9S144(15)
and, in pituitary adenoma, from D9S1749 to D9S199(17)
. These deleted regions that are defined in pituitary adenomas and in melanomas include both of the common regions of deletion in breast cancer that are described here. Moreover, one deleted region between D9S268 and D9S285 that has been identified in bladder cancer
(10)
overlaps with the region B identified in the present study. These findings suggest that the alterations of genetic material of distal 9p may be important for different human carcinomas. Additional investigations are underway to identify gene(s) involved in distal 9p alterations and to clarify their role in the pathogenesis of breast cancer and other types of human tumors.

Footnotes

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